miRNA regulation of angiogenesis: new roles for IGF-1R signaling and heparin

Allison Bierly, Technical and Marketing Writer
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microRNAs (miRNAs) regulate angiogenesis by manipulating the expression of pro- and anti-angiogenic factors and endothelial cell functions (1). Recently, new studies have yielded a wealth of discoveries about the action of these small, non-coding RNAs, revealing which miRNAs are involved in certain angiogenic settings, which signaling pathways they use to promote or inhibit angiogenic processes, and even how they can be delivered to other cells to modify cellular behavior. Some of the most striking findings include miR-126's regulon of pro-angiogenic factors (2), the role of the miR-23~24~27 cluster in choroidal neovascularization (3), and the clarification of how heparin blocks angiogenesis through modulation of miR-10b expression (4). Greater understanding of which miRNAs act in various angiogenic settings has the potential to introduce new miRNA-based therapies, such as mimics and antagomirs, into the realm of diseases such as cancer, ischemia, and retinopathy.
  • miR-126 silencing in breast cancer cells leads to pro-angiogenic signaling
  • Possible therapeutic roles of miR-24 and miR-132
  • miR-23 and miR-27 repress anti-angiogenic SPROUTY2 and SEMA6A
Tumor angiogenesis and metastasis - miR-126, miR-21, miR-320, and microvesicles
miR-23~27~24 in choroidal neovascularization/AMD and embryonic angiogenesis
miR-10b and heparin 
Targeting miR-24 and miR-132 for myocardial infarction
Back to top Introduction
The sprouting and development of blood vessels affects numerous processes in the body, and excessive or insufficient angiogenesis can exacerbate a variety of disease states. Therefore, precise regulation of angiogenesis is crucial to an organism's survival. Studies knocking down Dicer and Drosha implicated miRNAs in regulation of angiogenesis, and subsequent studies revealed roles for miR-126, the miR17~92 cluster, miR378, miR-210, miR-296, and others in various settings such as neoangiogenesis in response to injury, developmental angiogenesis, and tumor angiogenesis. (1, 5). Over the past year, significant progress has been made in discovering which miRNAs drive this process in both normal physiology and in various disease states. Due to these studies and others, a clearer picture of the miRNA network governing angiogenesis is starting to emerge. This review spans some of the most significant recent discoveries that have contributed to our understanding of tumor angiogenesís. 
Back to top Tumor angiogenesis and metastasis - miR-126, miR-21, miR-320, and microvesicles
Angiogenesis is a key component of tumor metastasis, and accordingly, miRNAs implicated in metastasis frequently regulate angiogenic processes such as endothelial cell recruitment. miR-126 is known as an angiogenesis regulator in development and neoangiogenesis after myocardial infarction (1). A recent study also uncovered a striking regulon controlled by this miRNA in metastasis, demonstrating that miR-126 targets 3 genes in order to modulate tumor angiogenesis, MERTK, IGFBP2, and PITPNC1 (2). The authors showed that silencing of miR-126 in breast cancer cells eases the repression on these genes, allowing cancer cells to trigger endothelial cell migration and chemotaxis via IGF-1 receptor signaling (see figure miR-126 silencing in cancer cells leads to angiogenesis through IGF1 receptor signaling) and the GAS6-MERTK pathway. Increased endothelial cell migration led to enhanced angiogenesis and metastatic colonization, defining a new set of mechanisms by which miR-126 silencing non-cell-autonomously contributes to cancer progression.

Conversely, miR-21 positively regulates angiogenesis through multiple signaling pathways, as described in a human prostate cancer cell line and chicken chorioallantoic membrane (CAM) angiogenesis model. Overexpression of this miRNA induces high expression of HIF-1alpha and VEGF, both of which promote angiogenesis. Indeed, cells transfected with miR-21 induced more branching of microvessels in the CAM assay. PTEN, which suppresses PI3K/AKT and MAPK signaling, is the target mediating these effects; repression of PTEN by miR-21 releases these pathways, which then lead to HIF-1alpha upregulation through AKT and ERK1/2 activation, triggering angiogenesis (6).

PTEN is also closely linked to miR-320 in negative regulation of angiogenesis; deletion of this protein in murine mammary stromal fibroblasts (MMFs) leads to downregulation of miR-320 and upregulation of its target ETS2. When PTEN-null MMFs or PTEN-null MMFs expressing miR-320 were co-injected with mouse mammary epithelial tumor cells into immunocompromised mice, co-injection with the miR-320-expressing cells led to less new blood vessel formation in the resulting tumors. Moreover, knocking down miR-320 in PTEN+/+ cells led to more blood vessel formation and tumor growth, demonstrating the importance of miR-320 in regulating angiogenesis. Conditioned media from miR-320-expressing PTEN-null MMFs also diminished endothelial cell tube formation compared to media from regular PTEN-null MMFs, and decreased endothelial cell proliferation (7).

In addition to being produced by a cell and modulating the expression of its own angiogenic or antiangiogenic factors, miRNAs may also trigger angiogenesis via delivery to endothelial cells in microvesicles (MVs) released by cancer stem cells (8). CD105+ mesenchymal renal cancer stem cells produce MVs expressing miRNAs that target genes involved in cell proliferation and adhesion, among other processes, in addition to mRNAs for growth factors and matrix metalloproteinases. Endothelial cells exposed to CD105+ MVs form capillary-like structures in vitro and display enhanced invasiveness, and both of these are diminished if the MVs are pretreated with RNase, suggesting that the RNA species are responsible for these effects. Intravenous injection of these MVs led to higher VEGF expression from lung epithelial cells, and endothelial cells treated with the MVs from cancer stem cells also showed enhanced angiogenic ability in Matrigel in vivo when injected into SCID mice.
Back to top miR-23~27~24 in choroidal neovascularization/AMD and embryonic angiogenesis
Choroidal neovascularization occurs in age-related macular degeneration, and a recent study suggested that angiogenesis in this setting may be controlled by the miR cluster 23~27~24 (3). When expression of these miRNAs was manipulated in several different contexts, angiogenesis was affected. miR-23 and miR-27 knockdown in endothelial cells cultured on Matrigel led to diminished capillary-like structure formation in vitro, and inhibition or overexpression with an ex vivo aortic ring model showed a similar relationship between these miRNAs and sprouting of the aortic ring cells. In vivo, locked nucleic acid (LNA) anti-miRs for miR-23 and miR-27 injected into mouse eyes at postnatal day 2 diminished angiogenesis, as measured 4 days later by sprouting distance and vascular coverage in the retina. Moreover, in a choroidal neovascularization (CNV) mouse model, the LNA-anti-miR treatment reduced CNV after laser injury by more than 50% compared to controls.

miR-23 and miR-27 exert their effects through targeting two genes whose products interfere with proangiogenic signaling, SPROUTY2 and SEMA6A (see figureThe miR23~27~24 cluster targets SEMA6A and SPROUTY2 to enhance VEGF receptor-mediated angiogenesis ). SEMA6A blocks VEGF receptor phosphorylation in response to its ligand, which affects downstream pro-angiogenic signaling pathways. Meanwhile, SPROUTY2 represses RAS/RAF/ERK signaling. Knockdown of miR-23 and miR-27 supports this suppression, leading to diminished endothelial cell sprouting. A new study has reinforced the miR-27/SEMA6A connection, demonstrating that overexpressing miR-27 enhanced sprouting by endothelial cells, while in vivo experiments in zebrafish showed that its inhibition diminished formation of embryonic vessels (9). SEMA6A silencing could rescue a portion of endothelial cell sprouting diminished by miR-27 inhibition, indicating that the effects of miR-27 on angiogenesis are partially executed through targeting of SEMA6A.
Back to top miR-10b
Interference with a miRNA, miR-10b, may also explain the antiangiogenic effects of the anticoagulant glycosaminoglycan heparin (4). miR-10b is pro-angiogenic; its overexpression in human microvascular endothelial cells (HMEC) enhanced capillary network formation in vitro and also led to more microvessel formation an in vivo model with Matrigel plugs compared to control cells. miR-10b is upregulated by thrombin, along with its transcription factor, Twist, and represses the translation of homeobox D10 (HoxD10). HoxD10 exerts antiangiogenic effects in endothelial cells, suppressing their migration as well as tube formation. Heparin binds to thrombin and blocks upregulation of miR-10b expression, thereby releasing HoxD10 regulation and preventing angiogenesis (see figure Heparin suppresses angiogenesis by blocking miR10b induction ).
Back to top Targeting miR-24 and miR-132 for myocardial infarction
 Insufficient angiogenesis is a major concern for myocardial infarction (MI), and miR-24 appears to play a role in exacerbating the problem. A study in mice recently showed increased levels of miR-24 in cardiac endothelial cells after ischemia, and further demonstrated that this miRNA targets GATA2 and PAK4 to promote endothelial cell apoptosis and prevent tube formation, a critical event in angiogenesis. Conversely, inhibiting miR-24 in cardiac endothelial cells using an antagomir in vivo actually promoted greater capillary density and restricted infarct size in mice following MI, suggesting that miR-24 may be an effective target for therapy to improve angiogenesis in this setting (10).

However, direct inhibition of anti-angiogenic miRNAs is not the only miRNA-based therapeutic option for myocardial infarction. Cell therapy using human pericyte progenitors in infarcted mouse heart led to improved repair through a number of mechanisms including enhanced angiogenesis (11). Dissection of the mechanism showed that the pericyte progenitor cells secreted VEGF-A and Ang-1, but also produced and secreted miR-132, a miRNA recognized for its role in angiogenesis in the context of cancer (12). The study found that, in vitro, conditioned medium from cultures of the pericyte progenitor cells triggered network formation and proliferation in endothelial cells. This was dependent on miR-132, as inhibition of this miRNA in the progenitor cells abolished these effects. In vivo, if miR-132 was inhibited, the pro-angiogenic effects of transplanted pericyte progenitors were also partially lost.
Back to top Conclusions
miRNAs are crucial regulators of many biological processes, and knowledge about their roles in angiogenesis has continued to expand. Due to the importance of angiogenesis for normal vascular development and homeostasis, as well as in disease processes like cancer, ischemia, and macular degeneration, miRNAs represent a significant biological target for future therapies.

Quantifying specific miRNAs and mRNAs in biological systems is a sensitive and reliable method for identifying gene expression changes as a result of disease states, development, or experimental perturbations. RT2 Profiler PCR Arrays and miScript miRNA PCR Arrays make this quantification swift, practical, and focused, uncovering the secrets of biological pathways and disease states for any laboratory with a real-time PCR instrument.
Back to top References
  1. Wang, S., and Olson, E.N. (2009) AngiomiRs – key regulators of angiogenesis. Curr. Opin. Genet. Dev. 19, 205.
  2. Png, K.J., Halberg, N., Yoshida, M., and Tavazoie, S.F. (2011) A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells. Nature 481, 190.
  3. Zhou, Q., Gallagher, R., Ufret-Vincenty, R., Li, X., Olson, E.N., and Wang, S. (2011) Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23~27~24 clusters. Proc. Natl. Acad. Sci. 108, 8287.
  4. Shen, X. et al. (2011) Heparin impairs angiogenesis through inhibition of microRNA-10b. J. Biol. Chem. 286, 26616.
  5. Biyashev, D., and Qin, G. (2011) E2F and microRNA regulation of angiogenesis. Am. J. Cardiovasc. Dis. 1, 110.
  6. Liu, L-Z. et al. (2011) miR-21 induced angiogenesis through AKT and ERK activation and HIF-1alpha expression. PLoS One 6, e19139.
  7. Bronisz, A. et al. (2012) Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320. Nat. Cell. Biol. 14, 159.
  8. Grange, C. et al. (2011) Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche. Cancer Res. 71, 5346.
  9. Urbich, C. et al. (2012) MicroRNA-27a/b controls endothelial cell repulsion and angiogenesis by targeting semaphorin 6A. Blood 119, 1607.
  10. Fiedler, J. et al. (2011) MicroRNA-24 regulates vascularity after myocardial infarction. Circulation 124, 720.
  11. Katare, R. et al. (2011) Transplantation of human pericyte progenitor cells improves the repair of infracted heart through activation of an angiogenic program involving micro-RNA-132. Circ. Res. 109, 894.
  12. Anand, S. et al. (2010) MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis. Nat. Med. 16, 909.

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